Supplemental Figures. B cell tolerance via GARP-TGF-β axis

Transcription

1 cell tolerance via GRP-TGF-β axis Supplemental Figures.. Spleen... Spleen mln Total FoxP + FoxP + Helios + FoxP + Helios - Total FoxP + FoxP + Helios + FoxP + Helios - Peyer s Small Intestine mln cell Small intestine cell Supplemental Figure : hanges in other immune compartments and increased mucosal cell proliferation in cell Lrrc deficient M chimera mice. Immune compartments were analyzed in M chimera mice months post M reconstitution. () Live Gr + b + granulocytic cells were detected in the spleen by flow, n= biological replicates. () Total FoxP + Tregs and Helios +/- Tregs were detected in the spleen (n=), mesenteric lymph nodes (mln; n=), Peyer s patch (n=), and small intestine lamina propria (n=-) by flow cytometry. () Immune cells were isolated from the mln and small intestine, and stained for 9 to detect cells and intracellularly for Ki7, followed by flow cytometry analysis (n=- and mice). () ctive and total TGF-β levels were measured by ELIS in and GRP M chimera mice months post M reconstitution. Statistics performed by two-tailed t-test; p<., error bars represent S..

2 cell tolerance via GRP-TGFβ axis Lupus-free. Lupus int Transitional I - -. Follicular + int.7 Marginal Zone GRP Lupus free NZM Lupus NZM Follicular Trans I Marginal Zone GRP Supplemental Figure : cell GRP in murine lupus models. () Representative flow plots of GRP and LP expression gated on live total splenic 9 + cells from NZM mice with (Lupus) and without (Lupus-free) active lupus and the quantification of the data (right), n=, representative of two independent experiments. Statistical analysis performed by two-tailed t-test, p<., error bars represent S.. () Flow plots depicting GRP and LP surface expression on live splenic 9 + follicular cells ( + int ), marginal zone cells ( - + ), and transitional I cells ( - - ) from lupus NZM mice and the quantification of the data (right), n=, representative of two independent experiments. Statistical analysis performed by one-way NOV with Tukey s test for multiple comparisons, p<., p<., error bars represent S..

3 cell tolerance via GRP-TGFβ axis week old mice TM inj i.p. Pristane inj. i.p. 7 N analysis Monitor mice h cre- Lrrc f/f cell gated. Weeks h cre- Lrrc f/f h cre+ Lrrc f/f. h cre+ Lrrc f/f GRP h cre- Lrrc f/f cre+ h Lrrc f/f Supplemental Figure : h-er cre Lrrc f/f cell specific GRP deficient mice have an increase in early onset PIL. Female and h-er cre Lrrc f/f cell GRP mice were injected with Tamoxifen for 7 days to delete GRP, (n=) and GRP (n=) mice. () Scheme of GRP deletion with Tamoxifen and subsequent pristane injection to induce autoimmunity. () Mice were bled days after Tamoxifen injection and peripheral blood was cultured with LPS for hours to induce GRP. ells were stained with 9, GRP, and LP and analyzed by flow cytometry to confirm GRP deletion in mice. ( and ) Two weeks after pristane injection, sera were collected and N was measured. Representative images shown in. ata are representative of two independent experiments. Statistical analysis performed by unpaired two-tailed t-test; p<., error bars represent S..

4 cell tolerance via GRP-TGFβ axis month months... p=.. F E Peyer s patch. G.... mln. Supplemental Figure : GRP overexpression dampens pristane-induced autoimmunity in mice. rtt GRP mice were fed doxycycline in the drinking water one week prior to a single intraperitoneal pristane injection. oxycycline treatment was continued throughout the course of the experiment for both GRP (n=7) and GRP (n=) mice. () IgG N levels were measured in the serum of pristane treated mice at month and months post pristane injection. () Five μm thick kidney sections were stained with anti-igg-fit to detect immunoglobulin deposition in the glomeruli. ata shown are the quantification; n=7 and n= (one mice died prior to analysis). () Immune cells were isolated from the spleen and percentages of cells (9 + ), germinal center cells (GL ), and Ki7 + cells from and mice were quantified. () MSs (Gr + b + ) in pristane treated and mice were quantified. (E) Peyer s patch and mesenteric lymph node (mln) immune cells were isolated and percentages of T cells and T cells were quantified. (F) ctive and total TGF-β levels were measured in the sera of and GRP mice pre- and post-pil by ELIS. (G) Soluble GRP (sgrp) was measured in the sera of and GRP mice pre- and post-pil by ELIS. n= and n= Pre-PIL; n=7 and n= post-pil. Statistical analysis performed by unpaired two-tailed t- test; p<., p<., p<., error bars represent S..

5 cell tolerance via GRP-TGFβ axis GRP TGF-βRII p Supplemental Figure : GRP overexpression reduces Peyer s patch cell cellularity and proliferation. rtt GRP and control mice were both fed doxycycline in the drinking water for months, n= and n=-. () Total number of Peyer s patches on the small intestine were counted. () Peyer s patches were isolated and percentage of 9 + cells was analyzed by flow cytometry. () Peyer s patch cells were stained intracellularly for Ki7 to assess homeostatic proliferation and analyzed by flow cytometry. Each dot is representative of a separate biological replicate; data is representative of independent experiments. (-) Statistical analysis performed by two-tailed t-test; p<., error bar represents S.. () 9 + cells were isolated from the spleen and Peyer s patches of oxycycline treated and GRP mice, followed by RN isolation and cn synthesis. qrt PR was utilized to detect differences in GRP, TGF-βRII and p transcript level, normalized to β actin. Representative data from independent experiments with biological replicates. Statistical analysis performed by one-way NOV with Tukey s test for multiple comparisons, p<., p<., p<., error bars represent S..